The work described here is designed to understand the mechanisms of pre- mRNA splicing. This is important for human health in so far as changes in splicing and RNA processing in general have been correlated with disease. Our goal is to identify and characterize the factors involved in pre-mRNA splicing with the hope that this information will be useful in the development of therapy options. We outline biochemical, cell biological, and genetic approaches designed to research this goal. To better understand the mechanisms we intend to continue to test the hypothesis that SR proteins alone can function to join exons together prior to splicing. For this we are using biochemical assays and electron microscopic analysis of SR protein bound pre-mRNA complexes. Previous studies show that disease can be associated with changes in nuclear structure. For this reason we intend to study the localization of splicing factors to active sites of gene transcription. Finally, determination of how cells regulate pre-mRNA splicing will be incomplete until all required pre-mRNA splicing factors are identified. Only a few splicing factors have been identified to date, and splicing is still carried out in crude nuclear extract. We isolated an antibody that has made it possible to isolate genes encoding more than half of the proteins present in purified splicing complexes. We intend to analyze structure and pre-mRNA splicing function of these proteins using standard biochemical approaches as well as using a gene inactivation approach in C. elegans.